Bio-Rad Trans-Blot® Plus Cell User Manual
Page 19
10. These variables will change total resistance and current readings:
•
Alterations to the buffer make-up, (e.g., addition of SDS or changes in ion
concentration due to the addition of acid or base to adjust the pH of the buffers)
•
Gel pH, ionic strength, and percentage of acrylamide, especially if the gel has
not been fully equilibrated
•
Volume of buffer (current increases when volume increases)
•
Transfer temperature (current increases when temperature increases)
•
Time during the transfer at which reading was taken (current normally increases
as the buffering capacity diminishes with progress of the run)
3.3 Buffer Formulations
All recipes in this section make 1 liter of buffer. A total of 15 liters is sufficient for
transfer and gel/cassette assembly for the Trans-Blot Plus cell. Scale up the following
recipes appropriately. Note that some buffers can be made as concentrated stock
solutions and diluted prior to use. Some buffers may also be purchased as pre-made
concentrated stock solutions.
Do not add acid or base to adjust the pH of the following buffers. Use only
analytical grade methanol because metallic contaminants in low-grade methanol will
plate onto the electrodes. Always add methanol or ethanol last to prevent
precipitation.
Note: Some pH meter electrodes will not provide correct measurements of the pH of
Tris buffers. If the pH of the buffer is incorrect, check that the pH meter electrode is
designed to work with Tris buffers. If the pH meter electrode functions properly for
Tris buffers and the pH is below 8.0, remake the buffer.
1. Buffers for SDS-PAGE gels
Towbin Buffer
This is a general purpose transfer buffer that was first described by Towbin
8
.
Towbin Buffer with 20 % Methanol
25 mM Tris, 192 mM glycine, 20% v/v methanol, pH 8.3
a) Using 10X Tris /glycine buffer (catalog #161-0734 for 1L bottles or
catalog # 161-0757 for 5L cube)
100 ml of 10X Tris /glycine buffer
700 ml of deionized water (dd H
2
O)
200 ml of methanol
b) Using dry reagents:
3.03 g Tris
14.4 g glycine
600 ml deionized water (dd H
2
O)
200 ml of methanol
add dd H
2
O to 1 liter
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